Antimicrobial and Antibiofilm Effects of Combinatorial Treatment Formulations of Anti-Inflammatory Drugs—Common Antibiotics against Pathogenic Bacteria

With the spread of multi-drug-resistant (MDR) bacteria and the lack of effective antibiotics to treat them, developing new therapeutic methods and strategies is essential. In this study, we evaluated the antibacterial and antibiofilm activity of different formulations composed of ibuprofen (IBP), acetylsalicylic acid (ASA), and dexamethasone sodium phosphate (DXP) in combination with ciprofloxacin (CIP), gentamicin (GEN), cefepime (FEP), imipenem (IPM), and meropenem (MEM) on clinical isolates of Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) as well as the transcription levels of biofilm-associated genes in the presence of sub-MICs of IBP, ASA, and DXP. The minimal inhibitory concentrations (MICs), minimal biofilm inhibitory concentrations (MBICs), and minimum biofilm eradication concentrations (MBECs) of CIP, GEN, FEP, IPM, and MEM with/without sub-MICs of IBP (200 µg/mL), ASA (200 µg/mL), and DXP (500 µg/mL) for the clinical isolates were determined by the microbroth dilution method. Quantitative real-time-PCR (qPCR) was used to determine the expression levels of biofilm-related genes, including icaA in S. aureus and algD in P. aeruginosa at sub-MICs of IBP, ASA, and DXP. All S. aureus isolates were methicillin-resistant S. aureus (MRSA), and all P. aeruginosa were resistant to carbapenems. IBP decreased the levels of MIC, MBIC, and MBEC for all antibiotic agents in both clinical isolates, except for FEP among P. aeruginosa isolates. In MRSA isolates, ASA decreased the MICs of GEN, FEP, and IPM and the MBICs of IPM and MEM. In P. aeruginosa, ASA decreased the MICs of FEP, IPM, and MEM, the MBICs of FEP and MEM, and the MBEC of FEP. DXP increased the MICs of CIP, GEN, and FEP, and the MBICs of CIP, GEN, and FEP among both clinical isolates. The MBECs of CIP and FEP for MRSA isolates and the MBECs of CIP, GEN, and MEM among P. aeruginosa isolates increased in the presence of DXP. IBP and ASA at 200 µg/mL significantly decreased the transcription level of algD in P. aeruginosa, and IBP significantly decreased the transcription level of icaA in S. aureus. DXP at 500 µg/mL significantly increased the expression levels of algD and icaA genes in S. aureus and P. aeruginosa isolates, respectively. Our findings showed that the formulations containing ASA and IBP have significant effects on decreasing the MIC, MBIC, and MBEC levels of some antibiotics and can down-regulate the expression of biofilm-related genes such as icaA and algD. Therefore, NSAIDs represent appropriate candidates for the design of new antibacterial and antibiofilm therapeutic formulations.


Introduction
With the increased antibiotic resistance among bacteria, we are getting closer to re-living the pre-antibiotic era. Moreover, the slow rate of current antibiotic discovery might lead society to experience, in the near future, a shortage of effective antibiotic treatments to treat bacterial infections caused by resistant microorganisms. Therefore, developing new approaches and strategies is essential for treating infections caused by resistant bacteria [1]. Several studies have shown the antimicrobial and antibiofilm activity of nonsteroidal anti-inflammatory drugs (NSAIDs) and we have different reports about the synergistic effects of NSAIDs with antibacterial agents [1,2]. Moreover, some studies have shown antagonistic interactions between antibiotics with anti-inflammatory drugs and an increase in the MICs of antibiotics agents as well as an increasing biofilm formation in the presence of anti-inflammatory drugs, especially corticosteroids such as dexamethasone [1,2]. Anti-inflammatory drugs are divided into two groups: corticosteroids and NSAIDs [3]. These drugs are widely used in combination with antibiotics to treat infections since they are administered to reduce inflammation, pain, and fever [3]. The mechanism of action of NSAIDs such as ibuprofen and acetylsalicylic acid (aspirin) depends on the inhibition of cyclooxygenase enzymes [3]. Corticosteroid drugs such as dexamethasone and betamethasone are synthetic analogs of glucocorticoids, which have anti-inflammatory and immunosuppressive functions and their effects depend on blocking receptors and genomic and nongenomic pathways [3]. Bacteria can develop resistance to antibiotics through the formation of biofilms, acquisition of resistance genes, and chromosomal mutations [4,5]. In addition to developing resistance to antibiotics, biofilm formation in bacteria is an important defense mechanism against the host immune system, leading to recurrent infections and increasing the pathogenicity of bacteria [6]. Moreover, there is a strong correlation between bacterial biofilm formation and the development of infective endocarditis, atherosclerosis, and urinary tract infections [7]. Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) are important opportunistic human pathogens capable of causing severe infections in hospital settings, especially in immunosuppressed patients [8,9]. Biofilm formation in these bacteria plays an important role in their pathogenicity and resistance to antibiotic agents [6]. The algD operon in P. aeruginosa encodes the main enzymes for alginate biosynthesis. Alginate is a homo-and hetero-polysaccharide that consists of β-D-mannuronate (M) and α-L-guluronate (G) units and plays an important role in biofilm formation in P. aeruginosa [5,6]. In S. aureus, the icaABCD operon plays an important role in biofilm formation [10]. It has been demonstrated that biofilm formation in Staphylococcus spp. is mediated by the ica operon. This operon consists of the four genes, icaA, icaD, icaB, and icaC, that appear to be negatively regulated by icaR [10].
Several studies have shown the antibacterial effects of NSAIDs such as ibuprofen, diclofenac, aspirin, and celecoxib against various bacteria such as Klebsiella pneumoniae, Escherichia coli, P. aeruginosa, and S. aureus [1,11]. NSAIDs, in addition to antibacterial effects in the combination of antibiotics, increase the susceptibility of bacteria to antibiotics agents [1]. Therefore, combining antibiotic agents with NSAIDs can be a strategy for treating bacterial infections and controlling inflammatory conditions. Imipenem, meropenem, cefepime, ciprofloxacin, and gentamicin are among the most widely used antibiotics in the treatment of various bacterial infections especially in hospital settings; therefore, due to the simultaneous use of these antibiotics agents with anti-inflammatory drugs in treating infections, it is essential to pay attention to the interactions between these drugs when administered simultaneously. Moreover, increasing our knowledge in this regard could help us to uncover new strategies and methods for treating infections. In the present study, we determined the antibacterial and antibiofilm effects of treatment formulations composed of commonly used NSAIDs including ibuprofen (IBP), aspirin (acetylsalicylic acid: ASA), and dexamethasone sodium phosphate (DXP) in combination with ciprofloxacin, gentamicin, cefepime, imipenem, and meropenem as current antimicrobial agents; moreover, we monitored the transcriptional levels of biofilm-involved genes, including icaA and algD in S. aureus and P. aeruginosa, respectively, at sub-MICs of IBP, ASA, and DXP.

Bacterial Isolates
Bacterial isolates, including S. aureus (n = 10) and P. aeruginosa (n = 10), were collected from various clinical samples in Afzalipour hospital in Kerman, Iran, and the San Bernardo Hospital in Monterrey, Nuevo León, Mexico. All bacteria were confirmed and identified by biochemical and standard microbiological tests [12][13][14].

DNA Extraction and Detection of mecA and icaADBC Operon in S. aureus Isolates and algD in P. aeruginosa
The total DNA of the isolates was extracted by the boiling methods previously described [16]. Briefly, 3-4 bacterial colonies from pure cultures of clinical isolates on brain heart infusion agar (BHI, Condalab Co., Madrid, Spain) were removed and suspended in 500 µL sterile DNAase and RNAase free water and boiled for 5 min and centrifuged at 10,000 rpm. Then, the supernatant was used as DNA template for amplification in the polymerase chain reaction (PCR) technique. The PCR method was used for the detection of mecA and the icaADBC operon in S. aureus and algD in P. aeruginosa isolates [13,17,18]. The PCR conditions and primer sequences used to detect mecA, icaADBC, and algD are presented in Table 1. A PCR technique was performed in a volume of 25 µL, containing the following: 12.5 µL of Taq DNA Polymerase Master Mix RED (Ampliqon, Co., Stenhuggervej, Denmark), 1 µL of DNA template, 0.25 µL of each forward and reverse primer (10 pM), and 11 µL sterile DNase and RNase free water. The PCR products were evaluated by electrophoresis on 1% agarose gel in 0.5 × TBE buffer (5.4 g Tris base, 2.75 g Boric acid, 2 mL 0.5 M EDTA, in 1 L sterile water) and stained by Green Viewer dye (Green Viewer, Parstous Biotechnology, Co., Mashhad, Iran), and a gel image was obtained by using a gel documentation system.

Biofilm Production Assay
For the biofilm formation assay among clinical isolates of S. aureus and P. aeruginosa, we used the microtiter method previously described by Stepanovic et al. using 96-well polystyrene microtiter sterile plates [19]. Briefly, clinical isolates were cultured on nutrient agar plates at 37 • C overnight, and 0.5 McFarland standard was prepared from each bacterial sample in phosphate-buffered saline (PBS). Then, 20 µL of bacterial suspension was mixed with 180 µL trypticase soy broth (TSB, Condalab Co., Madrid, Spain) supplemented with 1% glucose and added to sterile 96-well polystyrene microtiter plates. After incubation overnight at 37 • C, the microplates were carefully washed three times with sterile PBS, and then microplates were inverted to dry for 20 min at room temperature. For biofilm quantification, 200 µL of 2% safranin dye solution was added to the well for 40 min at room temperature and washed three times with sterile PBS. Safranin bound to the biofilm in each well was extracted with 200 µL of pure ethanol, and the absorbance of the extracted safranin was measured at 490 nm in an ELISA reader (BioTek, Agilent Technologies, Inc., Santa Clara, CA 95051, USA). Each assay was performed in triplicate. TSB + 1% glucose medium was used as a negative control to determine background optical density (OD). The cut-off ODs for biofilm formation were determined as the average OD of the negative control + 3 × standard deviation (SD) of the negative control. The OD value was calculated for each microtiter plate separately. OD > 4 × ODc was considered a strong biofilm formation; 2 × ODc < OD ≤ 4 × ODc was considered a moderate biofilm formation; ODc < OD ≤ 2 × ODc was considered a weak biofilm formation; and OD ≤ ODc was considered a nonbiofilm formation. Staphylococcus epidermidis RP62A (ATCC 35984) and Pseudomonas aeruginosa PAO1 were used as positive controls in biofilm formation assays.

Anti-Inflammatory Drugs Interference Experiments
To evaluate the possible effect of anti-inflammatory drugs, including ASA, IBP, and DXP, on the antibacterial and antibiofilm activity of antibiotic agents, we determined the MIC, minimum biofilm inhibitory concentration (MBIC), and minimum biofilm eradication concentration (MBEC) of each antibiotic agent including GEN, CIP, FEP, IPM, and MEM with/without sub-MIC ASA, IBP, and DXP. DMSO was used as a solvent for antiinflammatory drugs, and 10 mg/mL stock solutions of IBP, ASA, and DXP in DMSO were used to prepare working solutions of ASA (200 µg/mL), IBP (200 µg/mL), and DXP (500 µg/mL) in an MH broth containing DMSO (5%, vol/vol). MIC, MBIC, and MBEC experiments were performed in triplicate in Mueller Hinton Broth (MH Broth, Condalab, Co, Madrid, Spain), and MH broth with 5% DMSO without IBP, ASA, DXP, and antibiotics was used as a control for the experiment [20]. Staphylococcus epidermidis RP62A (ATCC 35984) and P. aeruginosa PAO1 were standard strains in interference experiments.  assays were performed by the broth microdilution method in 96-well polystyrene sterile plates with a flat-bottom microplate format according to CLSI recommendation. Briefly, a bacterial isolate suspension with an inoculum of 1 × 10 6 CFU/mL was diluted in Tryptic Soy Broth (TSB) (Condalab, Co, Madrid, Spain) plus 1% glucose with a serial dilution of antibiotic agents including GEN, CIP, FEP, IPM, and MEM (Sigma-Aldrich, Inc, St. Louis, MO 68178, USA), with/without a constant concentration of one of the sub-MICs of ASA (200 µg/mL), IBP (200 µg/mL), and DXP (500 µg/mL), and then incubated for 24 h at 37 • C. After incubation, MBIC was determined by crystal violet staining as the lowest concentration of antibiotic agents with/without anti-inflammatory drugs that resulted in an OD 600 difference at or below 10% of the mean of two positive growth-control well readings [21,22].

MBEC of Isolates to Antibiotics Agents with/without ASA, IBP, and DXP
In this step, the effects of each anti-inflammatory drug, along with GEN, CIP, FEP, IPM, and MEM on biofilm eradication were investigated. MBEC assay was performed as previously described [2,21,22]. Biofilms in clinical isolates of S. aureus and P. aeruginosa were formed overnight in TSB plus 1% glucose at 37 • C in nontreated 96-well polystyrene sterile flat-bottom plates. Biofilms were washed three times with sterile PBS buffer and exposed to different serial dilutions of antibacterial agents, including GEN, CIP, FEP, IPM, and MEM with/without sub-MICs of ASA (200 µg/mL), IBP (200 µg/mL), and DXP (500 µg/mL) in fresh Mueller Hinton Broth. Briefly, the antimicrobial drugs were diluted in fresh MHB, and 50 µL was dispensed in each biofilm in the wells. Stock solutions of ASA, IBP, and DXP were prepared in 5% DMSO and diluted in sterile MHB to reach concentrations of 400, 400, and 1000 µg/mL, respectively, and then 50µL of one of the anti-inflammatory drugs was added to each well with/without the serial dilution of antibiotic agents and plates were incubated for 18 h at 37 • C. The concentration of antibiotic agents with/without IBP, ASA, and DXP that eradicated the mature biofilm was considered the MBEC (Similar to MBIC determination in the Section 2.5.2).

Gene Expression Experiment
Quantitative real-time PCR (qPCR) was used to determine the transcription levels of icaA in S. aureus and algD in P. aeruginosa [23,24]. The expression levels of gyrB in S. aureus and rpoD in P. aeruginosa were used as reference genes for normalizing the transcriptional levels of icaA and algD as target genes in S. aureus and P. aeruginosa, respectively [23,24]. Biofilm gene expression was calculated in each bacterium without the presence (as a calibrator) of anti-inflammatory drugs and with the presence (as treated) of anti-inflammatory drugs. Briefly, clinical isolates were grown separately in MH Broth with ASA (200 µg/mL), IBP (200 µg/mL), and DXP (500 µg/mL) (used as treated isolates) and without anti-inflammatory drugs (used as calibrator isolates) by using a shaker incubator at 37 • C and 180 rpm to the log phase (optical density at 600 nm [OD 600] = 0.8-1) and then bacterial cells were collected by centrifugation at 12,000 rpm in 5 min. The total RNA of bacterial isolates was extracted with RNX-Plus (SINACLON, Co, Tehran, Iran) according to the manufacturer's recommendations. Then, RNase-Free DNase I enzyme (SINACLON, Co, Tehran, Iran) was used to eliminate DNA contaminations. Total RNA concentration was determined by a Lambda spectrophotometer (PCRmax Limited, Co., Staffordshire, UK), and cDNA synthesis was performed by the Easy cDNA Synthesis Kit (Parstous Biotechnology, Co., Mashhad, Iran), according to the manufacturer's recommendations. The qPCR experiment was performed in triplicate in the presence of negative and positive controls in a volume of 20 µL, containing the following: 10 µL of Taq DNA Polymerase Master Mix Green (Ampliqon, Co, Stenhuggervej, Denmark), 0.5 µL of cDNA template, 0.2 µL of each primer (10 pM), and 9.1 µL sterile DNase and RNase free water in the StepOnePlus Real-Time PCR System (ThermoFisher SCIENTIFIC, Applied Biosystems, Co., California, USA). qPCR conditions and primer sequences are presented in Table 1. Transcript levels of icaA and algD were determined relative to the reference genes, and results are expressed as mean values ± standard deviation using a two-sided Student's t-test and ANOVA tests by GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA) [23,24].

Statistical Analysis
GraphPad Prism 8 (GraphPad Software Inc., USA) was used for the statistical analysis of data and figure production. All data were first assessed for normality using a Kolmogorov-Smirnov test. The results were found to be normally distributed (p > 0.05 in the K-S test) and were analyzed using a one-way ANOVA test and expressed as mean values ± standard (mean ± SEM). Pairwise comparisons between groups were then made using Tukey's post hoc tests, where the main effect was seen in ANOVA tests. Data that were not normally distributed (p < 0.05 in the K-S test) were analyzed using a Kruskal-Wallis test. Where the main effect was seen in Kruskal-Wallis tests, pairwise comparisons between groups were made using Dunn's multiple comparisons test. In each case, p < 0.05 was considered statistically significant.

Results
Clinical isolates of S. aureus were resistant to penicillin, gentamicin, amikacin, erythromycin, ciprofloxacin, tetracycline, clindamycin, and trimethoprim/sulfamethoxazole and were sensitive to linezolid and vancomycin. S. aureus isolates were MRSA and were positive for mecA and the icaADBC operon. All clinical isolates of P. aeruginosa were resistant to meropenem, doripenem, imipenem, ciprofloxacin, piperacillin/tazobactam, gentamicin, aztreonam, cefepime, and ceftazidime and positive for algD. Both clinical isolates were considered biofilm producers according to the microtiter method results. The range of MIC for IBP was 1024-2048 µg/mL, and the MIC range for ASA was 2048-8192 µg/mL among both clinical isolates of S. aureus and P. aeruginosa (Supplementary Tables S1-S5). DXP had no antibacterial effect against the bacterial isolates. The MIC 50 , 90 , MBIC 50 , 90 , and MBEC 50 , 90 of antibiotic agents alone and combined with IBP, ASA, and DXP for both clinical isolates of S. aureus and P. aeruginosa are presented in Table 2. IBP at 200 µg/mL showed synergistic effects in combination with some antibiotic agents and among MRSA isolates, causing the MIC and MBIC levels to be reduced 4-8-fold for CIP, 2-16-fold for GEN, and 8-32-fold for IPM, MEM, and FEP. In MRSA isolates in the presence of ASA (200 µg/mL), we observed synergistic effects, and the MIC and MBIC were decreased 0-2-fold for CIP and GEN, 2-16-fold for IPM, 2-4-fold for MEM, and 2-8-fold for FEP. In contrast to the synergistic effects of IBP and ASA, in the presence of DXP among MRSA isolates, we observed antagonistic effects, and DXP increased MIC and MBIC levels 2-8-fold for CIP and FEP, 4-16-fold for GEN, and 0-2-fold for IPM, although they had no effect on the levels of MIC and MBIC for MEM. The level of MBEC among MRSA isolates was decreased 2-fold for CIP, GEN, and MEM, 2-4-fold for IPM, and 2-8-fold for FEP in the presence of IBP. MBEC levels in the presence of ASA were reduced 0-2-fold for CIP, GEN, IPM, and FEP among clinical isolates of S. aureus. ASA had no effect on the levels of MBEC for MEM among MRSA isolates. IBP at 200 µg/mL in combination with antibiotic agents showed synergistic effects among carbapenem-resistant P. aeruginosa and caused the MIC and MBIC levels to be reduced 2-4-fold for CIP, MEM, and FEP, 2-fold for GEN, and 2-8-fold for IPM. Moreover, we observed the synergistic effects and changed levels of MIC and MBIC for some antibiotic agents plus ASA among carbapenem-resistant P. aeruginosa isolates. In the presence of ASA at 200 µg/mL level, the MIC and MBIC decreased 0-4-fold for IPM, 2-4-fold for MEM, and 2-8-fold for FEP, and ASA had no effect on the levels of MIC and MBIC for CIP and GEN among carbapenem-resistant P. aeruginosa isolates. There were similar findings for MRSA isolates. DXP at 500 µg/mL increased MIC and MBIC 8-16-fold for CIP, 2-8-fold for GEN, 2-fold for IPM and MEM, and 2-4-fold for FEP in carbapenem-resistant P. aeruginosa isolates. The level of MBEC of the antibiotic agents was decreased 0-2-fold for CIP, IPM, and FEP in combination with IBP in carbapenem-resistant P. aeruginosa isolates, and IBP had no effect on the levels of MBEC for GEN and MEM. Moreover, ASA only reduced 2-fold the level of MBEC for FEP and could not decrease the levels of MBEC for CIP, GEN, IPM, and MEM among carbapenem-resistant P. aeruginosa isolates. MBEC levels in the presence of DXP were increased 4-fold for CIP and 0-2-fold for GEN and MEM, and we did not observe any change in MBEC level for IPM and FEP in the presence of DXP among carbapenem-resistant P. aeruginosa isolates. The MIC, MBIC, and MBEC level changes among MRSA and carbapenem-resistant P. aeruginosa isolates are presented in Tables 3 and 4 Figure 6). Transcriptional levels of algD in P. aeruginosa isolates in the presence of IBP and ASA were significantly decreased compared to the control group, and expression levels of algD significantly increased in the presence of DXP ( Figure 6).

Discussion
The resistance of bacteria to antibiotic agents is increasing worldwide. The spread multi-drug resistant (MDR) bacteria such as MRSA or carbapenem-resistant P. aerugino isolates is a global threat [25]. Anti-inflammatory drugs are commonly used in combin tion with antibiotics to control the systemic effects of infection [2]. Therefore, evaluatin therapeutic formulations that combine the effects of these drugs and antibiotic agents o bacteria can be important. Most studies on anti-inflammatory drugs' antimicrobial an antibiofilm activity have focused on nonclinical bacterial isolates [11]. In this study, w evaluated the effect of anti-inflammatory drugs, including IBP, ASA, and DXP, on th antibacterial and antibiofilm activity of CIP, GEN, IMP, MEM, and FEP on nonduplica clinical isolates of carbapenem-resistant P. aeruginosa (n = 10) and MRSA (n = 10). We al determined the transcriptional levels of biofilm-related genes, including icaA and algD sub-MICs of IBP (200 µ g/mL), ASA (200 µ g/mL), and DXP (500 µ g/mL) in clinical isolat of MRSA and carbapenem-resistant P. aeruginosa, respectively. There are different an inflammatory drugs, including nonsteroidal, such as aspirin, ibuprofen, diclofenac, an naproxen, and corticosteroidal anti-inflammatory drugs, such as dexamethasone, betam thasone, and hydrocortisone, that are commonly used to ameliorate fever and other sym toms of acute and chronic infections [3]. We describe here that IBP and ASA, combine with some common antibiotics including CIP, GEN, IMP, MEM, and FEP, had decreasin effects on MIC, MBIC, and MBEC. DXP had increasing effects on the MIC, MBIC, an MBEC of some of these antibiotic agents (Tables 2-4, Figures 1-5).
According to the reports, using a combination of anti-inflammatory drugs and an biotics to treat infections can have different effects on the MIC, MBIC, and MBEC of an biotic agents [2,11]. Corticosteroid drugs such as dexamethasone and betamethasone a synthetic analogs of glucocorticoids that have anti-inflammatory and immunosuppre sive functions [3]. Many reports show that steroid hormones increase the expression lev of virulence and biofilm-associated genes, efflux pump genes associated with MDR, an the rate of replication of bacteria [26,27]. The virulent mucoid biofilm phenotype in (500 µg/mL). Graphs were drawn based on fold changes in transcriptional levels of icaA and algD in clinical isolates of S. aureus and P. aeruginosa, respectively. Data are displayed as the mean ± standard error of the mean from 3 replicate experiments and were analyzed using the ANOVA test. *, statistical significance with p ≤ 0.05; **, statistical significance with p ≤ 0.01; ***, statistical significance with p ≤ 0.001; and ****, statistical significance with p ≤ 0.0001.

Discussion
The resistance of bacteria to antibiotic agents is increasing worldwide. The spread of multi-drug resistant (MDR) bacteria such as MRSA or carbapenem-resistant P. aeruginosa isolates is a global threat [25]. Anti-inflammatory drugs are commonly used in combination with antibiotics to control the systemic effects of infection [2]. Therefore, evaluating therapeutic formulations that combine the effects of these drugs and antibiotic agents on bacteria can be important. Most studies on anti-inflammatory drugs' antimicrobial and antibiofilm activity have focused on nonclinical bacterial isolates [11]. In this study, we evaluated the effect of anti-inflammatory drugs, including IBP, ASA, and DXP, on the antibacterial and antibiofilm activity of CIP, GEN, IMP, MEM, and FEP on nonduplicate clinical isolates of carbapenem-resistant P. aeruginosa (n = 10) and MRSA (n = 10). We also determined the transcriptional levels of biofilm-related genes, including icaA and algD in sub-MICs of IBP (200 µg/mL), ASA (200 µg/mL), and DXP (500 µg/mL) in clinical isolates of MRSA and carbapenem-resistant P. aeruginosa, respectively. There are different anti-inflammatory drugs, including nonsteroidal, such as aspirin, ibuprofen, diclofenac, and naproxen, and corticosteroidal anti-inflammatory drugs, such as dexamethasone, betamethasone, and hydrocortisone, that are commonly used to ameliorate fever and other symptoms of acute and chronic infections [3]. We describe here that IBP and ASA, combined with some common antibiotics including CIP, GEN, IMP, MEM, and FEP, had decreasing effects on MIC, MBIC, and MBEC. DXP had increasing effects on the MIC, MBIC, and MBEC of some of these antibiotic agents (Tables 2-4, Figures 1-5).
According to the reports, using a combination of anti-inflammatory drugs and antibiotics to treat infections can have different effects on the MIC, MBIC, and MBEC of antibiotic agents [2,11]. Corticosteroid drugs such as dexamethasone and betamethasone are synthetic analogs of glucocorticoids that have anti-inflammatory and immunosuppressive functions [3]. Many reports show that steroid hormones increase the expression level of virulence and biofilm-associated genes, efflux pump genes associated with MDR, and the rate of replication of bacteria [26,27]. The virulent mucoid biofilm phenotype in P. aeruginosa increased in the presence of estradiol [27]. Moreover, estradiol can downregulate genes involved in nucleotide metabolism and fatty acid biosynthesis and may be associated with enhanced survival and persistence in Chlamydia trachomatis [27]. In a study in 2014, it was shown that estradiol compounds increase the planktonic growth and the ability of Fusobacterium nucleatum to co-aggregate, and, in addition, it was also shown that polysaccharide production and biofilm formation in Prevotella intermedia was enhanced by estradiol in vitro [26]. The effects of using some antimicrobial agents in combination with corticosteroid drugs in vivo have been described. A study in 1996 showed that cloxacillin combined with DXP was more effective than cloxacillin alone in treating bacterial arthritis caused by S. aureus in Swiss mice [28]. DXP did not interfere with fluconazole in a murine model of cryptococcosis [29]. Other studies show that the combined use of hydrocortisone with mupirocin and methylprednisolone with imipenem is more effective than the antibiotics alone against eczema and atopic dermatitis by S. aureus and severe pneumonia in children, respectively [30,31]. However, some reports have shown the adverse effects on the antimicrobial and antibiofilm activity of antibiotics with corticosteroid drugs such as DXP. A study in 2017 reported that the corticosteroid anti-inflammatory drug DXP abrogates the activity of antimicrobial drugs, including gentamicin, chloramphenicol, oxacillin, ceftriaxone, and meropenem when combined in vitro against planktonic and microbial biofilms of S. aureus and P. aeruginosa [2]. Studies showed that combining DXP with ceftriaxone and vancomycin in a rabbit model of pneumococcal meningitis causes treatment failure [32,33]. These different effects of using DXP in combination with antibiotics may be due to the antibiotics' pharmacological properties, the type of bacteria (clinical or nonclinical isolates), and the possibility of DXP interference with the bacterial physiological processes. However, dexamethasone derivatives such as nitro-dexamethasone have recently been introduced, showing acceptable antimicrobial effects. These effects of nitro-dexamethasone have been attributed to the NO group, which can cause antibiofilm activity [34]. Moreover, it has been reported that topical steroids, including fluticasone, mometasone, and budesonide, directly reduce biofilm production and MBIC in vitro in S. aureus ATCC 25923 [35]. In this study, as in some other studies, DXP reduced the susceptibility of the isolates to CIP, GEN, and FEP. Interestingly, DXP had no significant effect on the levels of MIC and MBIC of IPM and MEM in both clinical isolates of S. aureus and P. aeruginosa. Therefore, our findings contrast with the report in 2017 that showed DXP could abrogate MEM activity against clinical isolates of S. aureus and P. aeruginosa [2]. These findings about IPM and MEM can be considered for their use in combination with DXP, although more studies are needed. However, the differences between our results and other reports may be due to the genotype and clone type of bacteria, antibiotic resistance mechanisms, and growth conditions among bacterial isolates. For example, it has been shown that biofilm production in methicillin-sensitive S. aureus isolates (MSSA) is usually associated with polysaccharide intercellular adhesin (PIA) or poly-N-acetyl-glucosamine (PNAG), whereas in MRSA isolates the formation of biofilms frequently depends more on the proteinaceous matrix [36][37][38]. Moreover, it has been shown that some clones of MRSA, such as USA300, produce thicker and stronger biofilms than other MRSA clones [38]. The antimicrobial effects of IBP on PAO1 differ from other strains of P. aeruginosa and Gram-negative bacteria such as Burkholderia spp [20]. In addition, the pH range and the type of medium are effective on the antimicrobial function of ibuprofen. For example, it has been reported that the MIC levels of IBP against S. aureus isolates are 40 to 80 µg/mL at pH 5 [39]. Similarly, it has been observed that growth suppression by IBP on S. aureus and Staphylococcus epidermidis occurred at concentrations greater than 150 µg/mL to 450 µg/mL at a pH of 7 [40]. However, the type of growth media can have effects on gene expression, metabolic functions, and other physiological growth conditions of bacteria [20].
Similar to other studies, our results show that ASA and IBP, in combination with some antibiotic agents, can reduce the MIC, MBIC, and MBEC. A few reports have shown the antibacterial and antifungal activity of IBP and its synergy with antibiotic agents. In 2017, it was reported that ASA, IBP, and diclofenac have antibacterial activity against Gram-positive and Gram-negative pathogenic bacteria such as MRSA and P. aeruginosa [1]. Moreover, a synergism effect between IBP/ASA in combination with cefuroxime and chloramphenicol was reported on S. aureus [1].
IBP has been shown to have antifungal activity in vitro against dermatophytes with MIC 5-40 µg/mL [39]. A study has demonstrated that combining IBP with fluconazole resulted in synergic activity against Candida spp., and MICs of fluconazole among the fluconazoleresistant strains decreased 2-128-fold in combination with IBP [41]. Recently, it has been shown that the morphogenesis and pathogenicity of fungi can be affected by cyclooxygenase (COX) inhibitors such as ASA, IBP, and indomethacin, combined with antifungal drugs [42]. Inhibitors of cyclooxygenase iso-enzymes such as ASA and diclofenac effectively decrease the germ tube formation of Candida albicans isolates [43]. Studies have shown that NSAID compounds can reduce the ability of biofilm formation in Candida spp., and the combination of NSAIDs with antifungal drugs can have synergistic effects [41,42]. These effects may be due to the inhibition of prostaglandin E2 (PGE2) due to the inhibition of COX by NSAIDs [42,43]. However, the mechanistic action of NSAIDs on bacteria is not clear.
One of the important points about the antimicrobial and antibiofilm activity of antiinflammatory drugs is to pay attention to the concentration of their antimicrobial function in vitro compared to their plasma concentration. This concentration in humans for ASA is from 50 to 200 µg/mL, which are doses with antimicrobial and antibiofilm activity against some microorganisms [44]. Moreover, it has been shown that the antiquorum sensing and antibiofilm activity of IBP against P. aeruginosa is related to rising drug concentrations [45].
During infection, biofilm formation by bacteria plays an important role in bacterial colonization, antibiotic resistance, and the immune system [10]. The production of exopolysaccharides or extracellular polymeric substances (EPS) is important in biofilm formation in bacteria [10]. In S. aureus, the major exopolysaccharide produced for biofilm formation is termed polysaccharide intercellular adhesion (PIA), also known as poly-N-acetyl-glucosamine (PNAG) [10]. The enzymes that synthesize the PIA/PNAG are encoded by the icaADBC operon [10]. Among the ica operon genes, icaA and icaD encode transmembrane proteins involved in oligomer synthesis for PNAG formation [10]. In this study, DXP increased the transcriptional level of icaA, from which it can be concluded that steroid compounds such as DXP increase the production of biofilms by increasing the expression of operon ica genes and thus reduce the susceptibility to some antibiotic agents in S. aureus. However, in this study ASA as an NSAID drug did not show significant changes in icaA expression, although IBP significantly decreased the transcriptional level of the icaA in S. aureus.
Alginate overproduction by increasing biofilm formation can protect P. aeruginosa from phagocytosis and antibiotic penetration [6]. algD is the main operon involved in biofilm formation in P. aeruginosa and encodes the main enzymes for alginate synthesis [5,6]. In the present study, DXP increased the transcriptional level of algD, from which it can be concluded that steroid compounds such as DXP increase the production of alginate or biofilm by increasing the expression of algD operon genes and thus reduce the susceptibility of P. aeruginosa isolates to antibiotic agents. However, ASA and IBP reduced the expression level of algD in the present study, which may explain the decrease in susceptibility of bacteria to various antibiotics in the presence of these drugs. Decreased expression of biofilm-associated genes, including icaA and algD in the presence of IBP and ASA, may explain the reduction in the MBIC of antibiotics agents among both clinical isolates, and, in some cases, this reduction in the MBIC level was more than the MIC (Tables 3 and 4). However, some studies have reported that NSAIDs can reduce antibiotic susceptibility and bacterial pathogenicity through different mechanisms. IBP potentially uncouples oxidative phosphorylation in bacteria and causes depletion in the intracellular ATP concentration in P. aeruginosa PAO1 [20]. Another study reported that IBP and ASA could bind to DNA gyrase and inhibit the growth of bacteria such as antibiotic agents. Recent chemoinformatics-and bioinformatics-based studies have shown that IBP has a similar structure to the quinolones and fluoroquinolones classes of antimicrobials [46]. In addition to the antibiofilm activity of NSAIDs against S. aureus, there is evidence pointing to the activity of NSAIDs against the pathogenicity of this bacterium by antivirulence properties such as the inhibition of hemolysis and staphyloxanthin production in this bacterium [47]. ASA has antibacterial and antibiofilm activity against S. aureus by blocking agrA-regulated virulence genes and down-regulating the expression of biofilm-associated genes such as icaA and fnbA. However, in our study, icaA expression was down-regulated in the presence of ASA but was not statistically significant [11]. Intercellular signaling, often known as quorum sensing (QS), is involved in biofilm development, and it has been shown that ASA can inhibit quorum sensing in P. aeruginosa [11,48]. A study demonstrated that ibuprofen could inhibit biofilm formation and quorum sensing in P. aeruginosa, which is probably related to the binding of ibuprofen to LuxR, LasR, LasI, and RhlR proteins [45].

Conclusions
This study shows that the antimicrobial and antibiofilm ability of some antibiotic agents increased in the presence of IBP and ASA as NSAIDs. Moreover, DXP reduces the susceptibility of bacteria to ciprofloxacin, gentamicin, and cefepime by increasing biofilm production, but it does not affect the activity of the imipenem and meropenem. This increase or decrease in the susceptibility of bacteria to antibiotic agents in the presence of the anti-inflammatory drug, in addition to their type and concentration, may be related to the type or clone of bacteria, physiological conditions of bacteria, and antibiotics class. With the increasing resistance to antibiotic agents among bacteria, proposing new strategies to treat infections and new antibacterial compounds is important. According to our findings, although more studies are needed, formulation treatments including NSAIDs such as ibuprofen and its derivatives alone or in combination with antibiotic agents can be suggested as antibiofilm compounds for treating bacterial-biofilm-associated infections. However, the data in this work show that dexamethasone, in interaction with antibiotics agents, may have adverse effects on their function, which is suggested to be considered in the treatment of infections. Finally, the results in this work highlight interactions between different antibiotics and common anti-inflammatory drugs and provide essential insights into the design and development of novel compounds or a new formulation strategy for treating bacterial infections.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pharmaceutics15010004/s1, Table S1. The MIC, MBIC, and MBEC to ciprofloxacin (µg/mL) alone and with sub-MIC of IBP, ASA, and DXP as anti-inflammatory drugs. Table S2. The MIC, MBIC, and MBEC to gentamicin (µg/mL) alone and with sub-MIC of IBP, ASA, and DXP as anti-inflammatory drugs. Table S3. The MIC, MBIC, and MBEC to imipenem (µg/mL) alone and with sub-MIC of IBP, ASA, and DXP as anti-inflammatory drugs. Table S4. The MIC, MBIC, and MBEC (µg/mL) to meropenem alone and with sub-MIC of IBP, ASA, and DXP as anti-inflammatory drugs. Table S5. The MIC, MBIC, and MBEC (µg/mL) to cefepime alone and with sub-MIC of IBP, ASA, and DXP as anti-inflammatory drugs.